A comparative evaluation of diffuse reflectance and Raman spectroscopy in the detection of cervical cancer

Optical spectroscopic techniques show improved diagnostic accuracy for non‐invasive detection of cervical cancers. In this study, sensitivity and specificity of two in vivo modalities, i.e diffuse reflectance spectroscopy (DRS) and Raman spectroscopy (RS), were compared by utilizing spectra recorded from the same sites (67 tumor (T), 22 normal cervix (C), and 57 normal vagina (V)). Data was analysed using principal component – linear discriminant analysis (PC‐LDA), and validated using leave‐one‐out‐cross‐validation (LOOCV). Sensitivity, specificity, positive predictive value and negative predictive value for classification between normal (N) and tumor (T) sites were 91%, 96%, 95% and 93%, respectively for RS and 85%, 95%, 93% and 88%, respectively for DRS. Even though DRS revealed slightly lower diagnostic accuracies, owing to its lower cost and portability, it was found to be more suited for cervical cancer screening in low resource settings. On the other hand, RS based devices could be ideal for screening patients with centralised facilities in developing countries.

     

Inside Back Cover: The conformation of bovine serum albumin adsorbed to the surface of single all‐dielectric nanoparticles following light‐induced heating (J. Biophotonics 7/2018)

Germanium vs Silicon: All‐dielectric nanoparticles provides the heat resistance for proteins under light‐induced heating. Further details can be found in the article by Andrei A. Krasilin et al. ( e201700322 )

     

Inside Back Cover: Multiphoton dynamic imaging of the effect of chronic hepatic diseases on hepatobiliary metabolism in vivo (J. Biophotonics 9/2018)

In vivo multiphoton imaging was used to map changes in hepatobiliary metabolism in liver fibrosis (left column) and hepatocellular carcinoma (right column). The top row shows the maps of kinetic rate constant of the uptake and esterase processing while the bottom row shows that of bile canalicular excretion of xenobiotics. Further details can be found in the article by Chih‐Ju Lin, Sheng‐Lin Lee, Wei‐Hsiang Wang, et al. ( e201700338 ).

     

Identification of ex‐vivo confocal scanning microscopic features and their histological correlates in human skin

Ex‐vivo confocal laser scanning microscopy (CLSM) is an emerging diagnostic tool allowing fast and easy microscopic tissue examination. The first generation of ex‐vivo devices have already shown promising results in the ex‐vivo evaluation of basal cell carcinoma compared to Mohs surgery. Nevertheless, for the diagnostics of pathological skin lesions the knowledge of normal skin features is essential. Therefore we examined 50 samples of healthy skin from various donor sites including head and neck (n = 25), trunk (n = 10), upper (n = 10) and lower extremities (n = 5) using a new generation ex‐vivo CLSM device offering three different laser wavelengths and compared the findings to the corresponding histological sections. In correlation with the histopathology we identified different layers of the epidermis, differentiated keratinocytes from melanocytes and described in detail skin appendages including hair follicle, sebaceous and sweat glands. Furthermore, structures of the dermis and subcutis were illustrated. Additionally, artefacts and pitfalls occurring with the use of ex‐vivo CLSM have been documented. The study offers an overview of the main ex‐vivo CLSM skin characteristics in comparison to the standard histological examination and helps to recognize and avoid common artefacts.

Anatomy of a hair follicle in the reflectance mode (RM) CLSM, fluorescence mode (FM) CLSM and in a routine hematoxylin‐eosin stained histological section (H).     

Front Cover: Photoluminescence intensity ratio of Eu‐conjugated lactates—A simple optical imaging technique for biomarker analysis for critical diseases (J. Biophotonics 5/2018)

Eu³⁺integrated photoluminescence intensity ratio (PLIR) approach for optical detection of lactates in blood serum, plasma and confocal imaging of brain tissues has very high potential for exploitation of this technique in both in vitro monitoring and in vivo bioimaging applications for the detection of biomarkers in various diseases states. This image is diagrammatic representation of fact that the overall PLIR is higher with more lactates conjugated with Eu³⁺ ions. Further details can be found in the article by Tarun Kakkar et al. ( e201700199 ).

     

In‐vivo multispectral video endoscopy towards in‐vivo hyperspectral video endoscopy

For in‐vivo diagnostics of cancer and pre‐cancer in the stomach, there is no endoscopic procedure offering both high sensitivity and high specificity. Our data suggest that multispectral or hyperspectral imaging may be helpful to solve this problem. It is successfully applied to the detection and analysis of easily reachable carcinomas, ex‐vivo samples of hollow organ mucosal carcinomas and also histological samples. An endoscopy system which allows flexible multispectral videoendoscopy for in‐vivo diagnostics has so far been unavailable. To overcome this problem, we modified a standard Olympus endoscopy system to conduct in‐vivo multispectral imaging of the upper GI tract. The pilot study is performed on 14 patients with adeno carcinomas in the stomach. For analysis, Support Vector Machine with linear and Gaussian Kernel, AdaBoost, RobustBoost and Random‐Forest‐walk are used and compared for the data classification with a leave‐one‐out strategy. The margin of the carcinoma for the training of the classifier is drawn by expert‐labeling. The cancer findings are cross‐checked by biopsies. We expect that the present study will help to improve the further development of hyperspectral endoscopy and to overcome some of the problems to be faced in this process.

     

Quantitative IR microscopy and spectromics open the way to 3D digital pathology

Currently, only mass‐spectrometry (MS) microscopy brings a quantitative analysis of chemical contents of tissue samples in 3D. Here, the reconstruction of a 3D quantitative chemical images of a biological tissue by FTIR spectro‐microscopy is reported. An automated curve‐fitting method is developed to extract all intense absorption bands constituting IR spectra. This innovation benefits from three critical features: (1) the correction of raw IR spectra to make them quantitatively comparable; (2) the automated and iterative data treatment allowing to transfer the IR‐absorption spectrum into a IR‐band spectrum; (3) the reconstruction of an 3D IR‐band matrix (x, y, z for voxel position and a 4^th dimension with all IR‐band parameters). Spectromics, which is a new method for exploiting spectral data for tissue metadata reconstruction, is proposed to further translate the related chemical information in 3D, as biochemical and anatomical tissue parameters. An example is given with oxidative stress distribution and the reconstruction of blood vessels in tissues. The requirements of IR microscopy instrumentation to propose 3D digital histology as a clinical routine technology is briefly discussed.

     

Classification of Gemcitabine resistant Cholangiocarcinoma cell lines using synchrotron FTIR microspectroscopy

Cholangiocarcinoma (CCA), a cancer of bile duct epithelium, is a major health problem in Thailand especially in the northeast. Overall treatment outcomes have not shown much improvement because the disease is usually detected at an advanced stage and often shows chemotherapeutic resistance. High‐throughput Fourier Transform Infrared (FTIR) microspectroscopy can be used for cell classification and has the potential to diagnose cancer and possibly predict chemo‐response. This study was aimed to differentiate gemcitabine‐sensitive and gemcitabine‐resistant induction in two CCA cell lines (KKU‐M139 and KKU‐M214) and xenograft tissues using synchrotron‐FTIR microspectroscopy. Partial Least Squares Discriminant Analysis (PLS‐DA) could discriminate between chemo‐sensitive and chemo‐resistant cells in the FTIR fingerprint spectral region (1800–1000 cm^–1) with more than 90% sensitivity and specificity. The chemo‐resistant and chemo‐sensitive phenotypes were different in protein (amide I, amide II), lipids (carbonyl group and CH₃ deformation) and phosphodiester from nucleic acids. Additionally, spectra from xenograft tissues showed similar results to the cell line study with marked differences between chemo‐resistant and chemo‐sensitive CCA tissues, and PLS‐DA could discriminate the chemotherapeutic response with 98% sensitivity and specificity. This is the first study to demonstrate the use of FTIR microspectroscopy to assess chemo‐response both in vitro and in vivo.

     

Intravital multiphoton photoconversion with a cell membrane dye

Photoconversion, an irreversible shift in a fluorophore emission spectrum after light exposure, is a powerful tool for marking cellular and subcellular compartments and tracking their dynamics in vivo. This paper reports on the photoconversion properties of Di‐8‐ANEPPS, a commercially available membrane dye. When illuminated with near‐infrared femtosecond laser pulses, Di‐8‐ANEPPS undergoes multiphoton photoconversion as indicated by the supralinear dependence of the conversion rate ρ_pc on the incident power (), and by the ability to photoconvert a thin optical section in a three‐dimensional matrix. The characteristic emission spectrum changed from red to blue, and ratiometric analysis on single cells in vitro revealed a 65‐fold increase in the blue to red wavelength ratio after photoconversion. The spectral shift is preserved in vivo for hours, making Di‐8‐ANEPPS a useful dye for intravital cell marking and tracking applications.

     

Front Cover: Spectrally encoded coherence tomography and reflectometry: Simultaneous en face and cross‐sectional imaging at 2 gigapixels per second (J. Biophotonics 4/2018)

SECTR is a novel multimodal imaging platform for combined volumetric optical coherence tomography (OCT) and en face spectrally encoded reflectometry (SER). The authors demonstrate three‐dimensional motion‐tracking with millisecond temporal and micron spatial resolution using complementary data from OCT and SER, and preliminary algorithms and results showing real‐time image aiming and multi‐volumetric mosaicking for reconstruction of wide‐field composites. The image shows a noninvasively imaged nine‐field mosaic of in vivo human retina and depth‐resolved visualization of tissue microstructures. Further details can be found in the article by Mohamed T. El‐Haddad, Ivan Bozic, and Yuankai K. Tao ( e201700268 )

     

Inside Back Cover: Development of a 3‐dimensional tissue lung phantom of a preterm infant for optical measurements of oxygen—Laser‐detector position considerations (J. Biophotonics 3/2018)

The picture depicts the different 3d‐printed organs, thorax, lungs, heart and bone. Assembled it is used as an optical phantom of a preterm infant for performing percutaneous optical measurements of the gas content in the lungs. In order to simulate the optical properties of the tissue, the heart and thorax can be filled with liquid phantoms, a mixture of Intralipid and Indian Ink. Further details can be found in the article by Jim Larsson et al. ( e201700097 ).

     

Inside Cover: Polarimetric imaging of biological tissues based on the indices of polarimetric purity (J. Biophotonics 4/2018)

The cover shows the image enhancement of biological tissues provided by the Indices of Polarimetric Purity (IPPs). By measuring the Mueller matrix of a biological sample, using an imaging polarimeter, the IPPs are calculated. They are polarimetric indicators providing further synthetization of depolarizing samples and leading to enhanced image contrast for some biological structures. Once the IPPs are calculated, a pseudo‐colouring technique is applied for higher visualization. Further details can be found in the article by Albert Van Eeckhout et al. ( e201700189 )

     

Front Cover: A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution (J. Biophotonics 2/2018)

A new type of high‐throughput imaging flow cytometer (>20 000 cells s^‐1) based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. FACED imaging flow cytometers enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. It critically empowers largescale and deep characterization of single cells and their heterogeneity with high statistical power— an ability to become increasingly critical in single‐cell analysis adopted in a wide range of biomedical and life‐science applications. Further details can be found in the article by Wenwei Yan et al. ( e201700178 )

     

A compact fiber‐optic probe‐based singlet oxygen luminescence detection system

This paper presents a novel compact fiberoptic based singlet oxygen near‐infrared luminescence probe coupled to an InGaAs/InP single photon avalanche diode (SPAD) detector. Patterned time gating of the single‐photon detector is used to limit unwanted dark counts and eliminate the strong photosensitizer luminescence background. Singlet oxygen luminescence detection at 1270 nm is confirmed through spectral filtering and lifetime fitting for Rose Bengal in water, and Photofrin in methanol as model photosensitizers. The overall performance, measured by the signal‐to‐noise ratio, improves by a factor of 50 over a previous system that used a fiberoptic‐coupled superconducting nanowire single‐photon detector. The effect of adding light scattering to the photosensitizer is also examined as a first step towards applications in tissue in vivo.

     

In vivo terahertz reflection imaging of human scars during and after the healing process

We use terahertz imaging to measure four human skin scars in vivo. Clear contrast between the refractive index of the scar and surrounding tissue was observed for all of the scars, despite some being difficult to see with the naked eye. Additionally, we monitored the healing process of a hypertrophic scar. We found that the contrast in the absorption coefficient became less prominent after a few months post‐injury, but that the contrast in the refractive index was still significant even months post‐injury. Our results demonstrate the capability of terahertz imaging to quantitatively measure subtle changes in skin properties and this may be useful for improving scar treatment and management.

     

Probe‐based confocal laser endomicroscopy (pCLE) – a new imaging technique for in situ localization of spermatozoa

In azoospermic patients, spermatozoa are routinely obtained by testicular sperm extraction (TESE). However, success rates of this technique are moderate, because the site of excision of testicular tissue is determined arbitrarily. Therefore the aim of this study was to establish probe‐based laser endomicroscopy (pCLE) a noval biomedical imaging technique, which provides the opportunity of non‐invasive, real‐time visualisation of tissue at histological resolution. Using pCLE we clearly visualized longitudinal and horizontal views of the tubuli seminiferi contorti and localized vital spermatozoa. Obtained images and real‐time videos were subsequently compared with confocal laser scanning microscopy (CLSM) of spermatozoa and tissues, respectively.

Comparative visualization of single native Confocal laser scanning microscopy (CLSM, left) and probe‐based laser endomicroscopy (pCLE, right) using Pro Flex^TM UltraMini O after staining with acriflavine.     

Effective delivery of porphyrazine photosensitizers to cancer cells by polymer brush nanocontainers

Efficient drug delivery can be assigned to tasks that attract the most acute attention of researchers in the field of anticancer drug design. We have reported the first case of using amphiphilic polymer brushes as nanocontainers for photosensitizer delivery to cancer cells. Regular graft‐copolymers of hydrophobic polyimides with hydrophilic polymethacrylic acid side chains were loaded with photosensitive dye tetra(4‐fluorophenyl)tetracyanoporphyrazine (Pz) providing a sufficiently stable homogeneous fraction of fluorescent Pz‐loaded nanoparticles with a size of 100–150 nm. Pz‐loaded polymer brushes were substantially more efficient for Pz delivery into cells compared with other types of particles examined, Pz‐polyethyleneglycol and Pz‐methylcellulose. In vivo, an efficient Pz delivery to tumor can also be expected since the Pz‐PB particle size is in the optimal range for passive targeting. Pz‐PB showed pronounced photodynamic activity, while, that is important, in the absence of irradiation the PB carrier itself was significantly less toxic than the dye itself.

Summing up, water‐soluble polymer brushes with polyimide backbones and polymethacrylic acid side chains can be regarded as a novel type of nanocontainers providing efficient intracellular drug delivery for photodynamic therapy of cancers.     

Back Cover: Joint tagging assisted fluctuation nanoscopy enables fast high‐density super‐resolution imaging (J. Biophotonics 9/2018)

Based on multicolor quantum dots (QDs) labeling, the joint tagging assisted super‐resolution radial fluctuation (JT‐SRRF) nanoscopy achieves high‐fidelity super‐resolution imaging of subcellular microtubules and fast live‐cell parallel tracking of cholera toxin subunit B (CTB) induced lipid clusters spatially distributed below the optical diffraction limit. This method paves the way for fast high‐density parallel tracking, which is especially beneficial for the investigation of the intensive dynamics in live‐cell applications. Further details can be found in the article by Zhiping Zeng, Jing Ma, Peng Xi, and Canhua Xu ( e201800020 ).

     

Physiological influence of silica on germinating pollen as shown by Raman spectroscopy

The process of silicification in plants and the biochemical effects of silica in plant tissues are largely unknown. To study the molecular changes occurring in growing cells that are exposed to higher than normal concentration of silicic acid, Raman spectra of germinating pollen grains of three species (Pinus nigra, Picea omorika, and Camellia japonica) were analyzed in a multivariate classification approach that takes into account the variation of biochemical composition due to species, plant tissue structure, and germination condition. The results of principal component analyses of the Raman spectra indicate differences in the utilization of stored lipids, a changed mobilization of storage carbohydrates in the pollen grain bodies, and altered composition and/or structure of cellulose of the developing pollen tube cell walls. These biochemical changes vary in the different species.

     

Oxygenation dynamics of sepsis patients undergoing far‐infrared intervention based on near‐infrared spectroscopy

Near‐infrared spectroscopy (NIRS; continuous wave type) is a noninvasive tool for detecting the relative change of oxyhemoglobin and deoxyhemoglobin. To make this change, intervention methods must be applied. This study determined the hemodynamics of 44 healthy participants and 35 patients with sepsis during exposure to FIR as a novel physical intervention approach. Local microcirculation of their brachioradialis was monitored during exposure and recovery through NIRS. The variations in blood flow and microvascular reaction were determined by conducting paired and unpaired t tests. The oxyhemoglobin levels of the healthy participants increased continuously, even during recovery. In contrast to expextations, the oxyhemoglobin levels of the patients plateaued after only 5 min of FIR illumination. The proposed method has potential applications for ensuring efficient treatment and facilitating doctors in diagnosing the functions of vessels in intensive care units.

Mapping diagrams of HbO₂ in healthy males and males with sepsis illustrated unique scenarios during the process.